Creatine kinase (CK) occurs in animal body fluids and tissue in the form of three known isoenzymes, designated CK-BB, CK-MM and CK-MB. Each of these three isoenzymes, namely CK-BB, CK-MM, and CK-MB, differs one from the other by virtue of containing a different combination of subunits designated M or B. CK-BB has two B subunits, CK-MM has two M subunits, and CK-MB has one M and one B subunit. Determining the presence of CK-MB in biological fluids, especially in a patient's serum, has become very useful in the diagnosis of myocardial infarction. (Galen, RS., Human Path 6: No. 2, 145-147, Apr. 1975).
Current immunological methods for determining the presence of CK-MB and the disadvantages of such methods have recently been reported and reviewed (Current Problems in Cardiology, Vol. III, No. 12, March, 1979 p. 7-28). These methods have not been very satisfactory in resolving the difficulty of interference from CK-MM and CK-BB in distinguishing or determining CK-MB from CK-MM and CK-BB.
Immunological process for determining CK-MB activity in a sample of a biological fluid has been disclosed in U.S. Pat. Nos. 4,067,775 and 4,237,044. This disclosed process of these patents employs an antibody produced from an activated CK-MM antigen. In this process it is required that the fluid sample tested must not contain any CK-BB isoenzyme. In particularly U.S. Pat. No. 4,067,775 states in column 3 at lines 38-40 that "CK-BB interferes with the process of the invention and therefore must not be present in the biological fluids being tested". Since biological fluids contain CK-BB, the assay of these patents may require time consuming and difficult procedures to first remove any CK-BB from the fluid. If any CK-BB is in the fluid sample, false positive results would be produced through the use of this assay. Thus, this process cannot be employed to determine CK-MB in fluids containing CK-BB.
Wreton and Pfeiderer, Clinica Chimica Acta 58, 223-232 (1975) discloses an immunoassay system wherein CK-MM and CK-BB are determined by immunoinhibition and immunotitration assays. These assays require the quantitation of values obtained by comparing residual isoenzyme activity with total isoenzyme activity of the sample. An immunoinhibition assay for CK isoenzymes is one wherein the enzymatic activites of CK-isoenzymes are differentially inhibited or inactivated within a test sample immunologically by inhibiting or inactivating antibodies which maintain the homogeneity of the sample. The Wreton and Pfleiderer process requires a multiplicity of assays (at least four) to arrive at values needed to determine the CK-isoenzyme activities. There would, therefore, be correspondingly four sources of errors, along with an equal number of time consuming procedures.
A process disclosed by Wursburg et al. in J. Clin. Chem. Clin. Biochem. 15, 131-137 (1977) employs precipitating antibodies to differentiate CK-isoenzymes in a scheme which necessitates measurement from four different assays. The assays provide for measuring total CK activity in the test sample and residual activities after adding precipitating anti-CK-BB, precipitating unit-CK-MM and both of these precipitating antibodies to separate reaction vessels. By this process, homogeneity of the sample is not maintained by the precipitating antibodies. Like the Wreton-Pfleiderer process, this process requires laborous and time consuming multiple assays with correspondingly high sources of errors.